The objectives of this application are two-fold: 1. The recognition and processing of mismatched DNA base pairs contributes to the fidelity of chromosome replication and may also be responsible for certain marker effects in recombination. We have developed a cell-free E. coli system which supports methyl-directed mismatch correction in vitro, and have obtained all the proteins known to be required for this process (products of mutH, mutL, mutS, and uvrD genes) in near pure form. Since these proteins are not sufficient to mediate the reaction, we will isolate other required components with the aim of reconstituting the reaction in a defined system. This is a prerequisite for a major aim of the work, namely to establish the biochemical mechanism by which this complex reaction occurs. Our second goal with this system is to exploit its ability to recognize mispaired bases in attempts to develop a reagent system for the detection and localization of point mutations in vitro. 2. The second major objective of the proposed work is to further elucidate the molecular mechanisms involved in specific DNA sequence recognition and catalysis by EcoRI restriction and modification enzymes. Our efforts in this respect will be along two lines. Utilizing kinetic and binding methods, we will try to further delineate the mechanisms of DNA search and site-specific catalysis by these enzymes. Secondly, we will continue ongoing molecular genetic experiments, the aim of which is to relate particular amino acid residues to functional activities of the two proteins.